The long-term objectives of this work are to identify and characterize genes that may play important roles in both normal and malignant hematopoiesis. Specifically, we are interested in defining regulators of hematopoietic stem cell (HSC) self-renewal versus differentiation cell fate decisions. HSCs are potentially useful for bone marrow (BM) transplantation for genetic disease and cancer, as targets for gene therapy, and as precursors for generation of blood cell products in vitro. Ex vivo expansion of HSCs that maintain primitive properties and functional activity continues to be an important research objective, considering the number of potential applications for use of HSCs. Growth factor signal transduction pathways are known to affect HSC differentiation along specific lineages by acting as survival factors to prevent apoptosis and by imparting a proliferative signal. Additional modulation of interactions of HSCs with the BM stromal microenvironment can be caused by growth factor stimulation. Our goal for this project is to determine the requirement for the JAKISTAT signal transduction pathway in the response of HSCs to specific growth factors that can activate STATs and also maintain HSC activity during short-term ex vivo culture. Multiple signal transduction pathways could be utilized in HSCs following ligand binding to the cell surface receptor and therefore these pathways could be redundant. For this reason, the role of STATs in primitive levels of hematopoiesis cannot be predicted from biochemical analysis of differentiated cells. Our initial studies have revealed that stem cells from mice lacking STAT5a and STAT5b expression show a marked deficiency in competitive long-term repopulating activity without a reduction in the absolute stem cell number. However, it needs to be determined whether the defect is related to cell cycle induction, apoptosis, or alteration in homing/engraftment ability. We propose to utilize molecular genetic approaches such as retroviral-mediated gene transfer and homozygous gene disruption in mice to characterize and distinguish the functional roles of STAT3 and STAT5 in hematopoietic stem cell function. The following specific aims will be addressed in this study. 1) We will determine the role of STAT5 in growth factor signaling pathways that are active in hematopoietic stem cells. 2) We will determine the role of STAT3 in growth factor signaling pathways that are active in hematopoietic stem cells. Altogether, this work will provide insight into the basic biology of HSCs and perhaps ultimately lead to novel methods for expansion of transplantable stem cells in vitro for therapeutic approaches that could be used clinically.